Cellular damage to aerobic organ tissues is well recognized as a consequence of prolonged ischemia, whether endogenous as in the case of a spontaneous coronary artery occlusion, or iatrogenic such as with open heart, coronary bypass surgery, or similar transplant procedures with other organs such as the lung, liver, kidney, and gastrointestinal tract. The degree and duration of the ischemia causing event are relevant to the amount of cell death and/or reversible cellular dysfunction. It is also known that much of the tissue damage, in fact, occurs upon reperfusion (i.e., resumption of blood flow) and re-oxygenation of the previously anoxic tissue. The phenomenon, referred to as reperfusion injury, has been the subject of considerable recent study prompted by medical advances particularly in the treatment of myocardial events such as myocardial infarction (MI) and other remedial procedures such as coronary bypass, other open heart surgeries, and organ transplants.
As a side product of normal aerobic respiration, electrons are routinely lost from the mitochondrial electron transport chain. Such electrons can react with molecular oxygen to generate the reactive free radical superoxide which through other reaction steps in the presence of hydrogen peroxide and iron produces the extraordinarily reactive and toxic hydroxyl radical. Metabolically active aerobic tissues possess defense mechanisms dedicated to degrading toxic free radicals before these reactive oxygen species can interact with cellular organelles, enzymes, or DNA, the consequences of which could, without such protective mechanisms, be cell death. These defense mechanisms include the enzymes superoxide dismutase (SOD) which disproportionates superoxide, catalase which degrades hydrogen peroxide, and the peptide glutathione which is a non-specific free radical scavenger.
While not fully understood, it is believed that with prolonged ischemia of metabolic tissues (twenty minutes or more) and subsequent reperfusion, a complex group of events occurs. Initially during the ischemic period, intracellular anti-oxidant enzyme activity appears to decrease, including that of SOD, catalase, and glutathione. There is also an indication that the level of xanthine oxidase activity concomitantly increases in vascular endothelial tissue during the ischemic event. The combination of enhanced ability to produce oxygen free radicals (via enhanced xanthine oxidase activity) and reduced ability to scavenge the same oxygen radicals (via reduced SOD, catalase and glutathione activity) greatly sensitizes the ischemic cell to an oxidative burst, and hence damage, should these cells be subsequently reperfused with blood and therefore oxygen. This oxidative burst occurring within seconds to minutes of reperfusion could result in reversible and irreversible damage to endothelial cells and other cells constituting the ischemic-reperfused organ matrix. If, for example, the heart is the organ under consideration, reversible oxidative damage can contribute to myocardial stunning, whereas irreversible damage presents itself as a myocardial infarction. Attendant with this initial oxidative burst is oxidation damage to cell membranes. Lipid oxidation in cell membranes appears to play a role in neutrophil chemotaxis to post-ischemic areas. Such activated neutrophils adhere to vascular endothelium, induce the conversion of xanthine dehydrogenase to xanthine oxidase within said endothelial cells, and further aggravate loss of endothelial integrity. Activated neutrophils also migrate out of the vasculature into myocardial interstitial spaces where the inflammatory cells can directly kill myocytes. Additionally, perturbations in normal calcium mobilization from sarcoplasmic reticulum as a consequence of ischemia-reperfusion contribute to reversible myocardial dysfunction referred to as myocardial stunning.
"Stunning" in lay terms refers to decreased, yet reversible, pump efficiency in the case of the heart which leads to decreased cardiac output and, hence, the symptomatology of suboptimal organ perfusion. Reperfusion of ischemic myocardial tissue may also cause electrophysiologic changes, including potentially lethal arrhythmias.
Therefore, the consequences of ischemia-reperfusion events are reversible and irreversible cell damage, cell death, and decreased organ functional efficiency. More specifically, in the case of myocardial reperfusion injury, the consequences include myocardial stunning, arrhythmias, and infarction, and as a sequela, cariogenic shock and potentially congestive heart failure.
The paradox of cellular damage associated with a limited period of ischemic anoxia followed by reperfusion is that cell damage and death appear not only likely to directly result from the period of oxygen deprivation but, additionally, as a consequence of re-oxygenation of tissues rendered highly sensitive to oxidative damage during the ischemic period. Reperfusion damage begins with the initial oxidative burst immediately upon reflow and continues to worsen over a number of hours as inflammatory processes develop in the same post-ischemic tissues. Efforts dedicated to decreasing sensitivity of post-anoxic cells to oxidative damage and, additionally, efforts to reduce inflammatory responses in these same tissues have been shown to reduce the reversible and irreversible damage to post-anoxic reperfused organs. A combination of methods to reduce both the initial oxidative burst and subsequent inflammation associated damage could provide synergistic protection against reperfusion injury.
With respect to the treatment of MI patients, a common therapy now used is to employ thrombolytics such as streptokinase and t-PA. U.S. Pat. No. 4,976,959 discloses the administration of t-PA and SOD to inhibit tissue damage during reperfusion and/or percutaneous transluminal coronary angioplasty during heart attacks to restore regional blood flow. Thus, an increasing number of patients are being exposed to the likelihood of reperfusion injury and its effects, particularly cardiac patients.
While exogenously administered, SOD is known to destroy free hydroxyl radicals and, therefore, in theory helps alleviate the oxidative burst which occurs upon reperfusion, in fact it does not have broad clinical applicability, has limited effectiveness, and to date has been restricted in its application to amelioration of ischemia-reperfusion injury involving the heart.
There is, therefore, a continuing need for the development of ischemia reperfusion-injury attenuating treatments which have broader applicability and greater effectiveness. For example, there are many ischemic reperfusion (re-oxygenation) events besides MI where an effective reperfusion injury protective agent could be utilized. Such events include organ transplants, traumatic limb amputation and reattachment, CNS trauma, Reye's syndrome, gut infarct, and other cardiac surgical procedures such as bypass surgery, value replacement, septal defect repairs.
Reperfusion injury to organs other than the heart will generally manifest itself in substantially reduced efficiency of function, a consequence of which may be premature degeneration of the organ. Additionally, transplanted organs experience enhanced rejection rates if there is significant underlying reperfusion injury.
As discussed briefly above, while the precise mechanism of reperfusion injury has not been clearly defined, mounting data, most of which has been gathered in various cardiac model studies, indicate that the generation of oxygen-derived free radicals, including superoxide anion (O.sub.2).sub.1, the hydroxyl free radical (.sup.-- OH) and H.sub.2 O.sub.2, results as a consequence of the reintroduction of molecular oxygen with reperfusion and plays an important role in tissue necrosis. Agents which either decrease the production of these oxygen derived free radicals (including allopurinol and deferroxamine) or increase the catabolism of these materials such as superoxide dismutase, catalase, glutathione, and copper complexes, appear to limit infarct size and also may enhance recovery of left ventricular function from cardiac stunning.
Brown et al. conducted experiments whereby it was shown that hearts isolated from rats pretreated with endotoxin twenty-four hours prior to ischemia and reperfusion showed increased myocardial catalase activity with consequent increased myocardial function as assessed by measurement of ventricular developed pressure contractility (+dp/dt) and relaxation rate (-dp/dt) compared to control hearts, as evidence of decreased injury. (See Proc. Natl. Acad. Sci. USA, Vol. 86, pp 2516-2520, April 1989, Physiological Sciences). (See also Bensard, et al., Endogenous Tissue Antioxidant Enzyme Activity, Journal of Surgical Research, Vol. 48, No. 6, June 1990.)
Berg et al. noted that rats pretreated with endotoxin were protected against lung injury during hyperoxia by pretreatment with endotoxin. They observed increased levels of tumor necrosis factor (TNF) and interleukin-1 (IL-1) in sera. (See J. Appl. Physiyol. 68(2): 549-553, 1990.)
Notwithstanding observations of the protective effect of endotoxin as discussed above, Smith conducted experiments to compare the protective effects of endotoxin, diphosphoryl lipid and monophosphoryl lipid A against lethal intrathoracic edema produced by continuous exposure to hyperoxia conditions. Sprague-Dawley rats were pretreated with one of the materials 72 hours prior to hyperoxia exposure. It was observed that toxic endotoxin and toxic diphosphoryl lipid A protected the rats against oxygen toxicity, but that non-toxic monophosphoryl lipid A actually potentiated pulmonary oxygen toxicity. (See Research Communications in Chemical Pathology and Pharmacology, Vol. 62, No. 2, Nov. 1988).
A variety of agents have been identified through preclinical investigations which appear to have the potential to provide some benefit in respect to reperfusion injury. Unfortunately, when applied to the human clinical setting, the results have been disappointing. SOD performed poorly at reducing myocardial infarct size in the clinical setting. A short half-life, poor tissue distribution, and consequently primarily a protective effect on vascular endothelium restricted clinical utility. Efforts to improve the half-life of SOD by forming the polyethylene glycol conjugate did not improve protective activity. Allopurinol has demonstrated some efficacy in early human studies involving renal transplants. Other agents entering human clinical trials include a monoclonal antibody to the neutrophil adhesion molecule CD18, a complement receptor antagonist, fluorinated hydrocarbons, and adenosine or adenosine agonists. However, these and other therapies under consideration have yet to exhibit the desired clinically useful attributes.
The pathogenesis of reperfusion injury is very complex, including depletion of anti-oxidant enzymes, alterations in xanthine oxidase activity, alterations in calcium mobilization, activation, chemotaxis and localization of inflammatory cells (neutrophils), and alterations in vascular permeability. Thus, prior to the present invention effective amelioration of reperfusion injury may require the use of multiple therapeutic agents each one of which acts to modify only one or at best a few or the aspects of pathogenesis. SOD or catalase administration supplements the depletion of these single enzymes. Allopurinol is a xanthine oxidase inhibitor. Adenosine restores ATP levels in post-ischemic tissue. Monoclonal antibodies to CD18 and complement receptor antagonist block neutrophil adhesion to vascular endothelium. The present invention which interferes with multiple aspects of reperfusion injury represents a substantial advance in the art.
It can therefore be seen that there is a need for a safe, effective composition having broad applicability to prevent or ameliorate the harmful effects of reperfusion with minimal side effects. It is a primary object of the present invention to fulfill this need.
Another object of the present invention is to provide a method for providing a high degree of protection for warm blooded animals against the harmful effects of reperfusion after a prolonged period of ischemia without the side effects attendant with therapies presently available.
Still another objective of the present invention is to provide a pharmaceutically acceptable carrier composition which can be used for intravenous administration of the compositions of the present invention without any significant undesirable side effects and without adversely affecting antigenic or immune stimulating properties.
These and other objects and benefits of the present invention will be apparent to those skilled in the art from the further description and accompanying claims.